Ultrastable antibody ionic liquids

ABSTRACT

A stable protein ionic liquid, comprising an anti-hemoglobin cation/anion pair. The anti-hemoglobin cation/anion pair may be an anionic polymer of poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether. The anti-hemoglobin cation/anion pair may further comprise a cationized anti-hemoglobin antibody, single-chain antibodies from camelids, antibody fragments, polyclonal Anti-horse spleen ferritin antibodies, monoclonal Anti-Flag antibodies, monoclonal Anti-HRP2 to Plasmodium falciparum, polyclonal Anti-neuropeptide Y, polyclonal Anti-human troponin, isotypes of antibodies, or combinations of multiple antibodies.

RIGHTS OF THE GOVERNMENT

The invention described herein may be manufactured and used by or forthe Government of the United States for all governmental purposeswithout the payment of any royalty.

Pursuant to 37 C.F.R. § 1.78(a)(4), this application claims the benefitof and priority to prior filed Non-Provisional application Ser. No.15/440,832, filed 23 Feb. 2017, now U.S. Pat. No. 10,463,733, andProvisional Application Ser. No. 62/403,774, filed 4 Oct. 2016, whichare expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to ultra-stable, water-freebiological materials and, more particularly, to ultra-stable,heat-resistant, biologically active, water-free protein ionic liquidsthat do not require refrigeration.

BACKGROUND OF THE INVENTION

Most biological materials (i.e. proteins and antibodies) thrive inaqueous environments and physiological conditions (neutral pH—between6-8, ambient temperatures 25-37° C.) in order to perform theirbiological function. Water is used for stabilizing some biomolecularstructures through hydrogen bonding, providing proton donors/acceptors,regulating binding interactions, and controlling molecular dynamics.Conversely, water is also detrimental to biomolecular structure andfunction by increasing the rate of hydrolysis and oxidation,destabilizing protein structure, and increasing thesusceptibility/sensitivity to elevated temperatures. In total, thisresults in denaturation, proteolytic degradation, decomposition, andshort shelf-lives.

In order to counteract the effects of water and limit decomposition,current biomolecules, e.g. proteins and antibodies, may require constantrefrigeration during storage, handling, and transport in order topreserve structure, functionality, and biological activity. Generally,antibodies in water may be stable for up to one month when stored atabout 4° C. and up to one year when stored in 25% glycerol at −20° C.The presence of water in a biological solution will typically result inhydrolysis, even if the temperature is reduced or the solution isfrozen. Water promotes hydrogen bonding, intramolecular interactions,stabilizes the antibody structure, facilitates mass transport anddiffusion of products, and regulates binding interactions. Water alsoincreases the sensitivity of the antibodies to elevated temperatures,destabilizes protein structures, increases hydrolysis and oxidationrates, reduces shelf lives, and promotes unfolding/denaturation.Consequently, the exclusion of water from antibody preparations ishighly appealing and offers a means towards reducing proteindegradation, increasing stability, enabling refrigeration-free storageand handling, and significantly increasing shelf-lives. In addition,even if freezing or refrigeration are acceptable alternatives, manyplaces around the world have no available electricity to powerrefrigeration equipment. The half-life of unrefrigerated antibodies maybe as short as 2 days.

SUMMARY OF THE INVENTION

The present invention overcomes the foregoing problems and othershortcomings, drawbacks, and challenges of making stablebiologically-active materials, such as proteins and antibodies. Whilethe invention will be described in connection with certain embodiments,it will be understood that the invention is not limited to theseembodiments. To the contrary, this invention includes all alternatives,modifications, and equivalents as may be included within the spirit andscope of the present invention.

Based on the numerous drawbacks associated with water in antibodysolutions, see above, the aim of this invention, in one embodiment, isthe removal of most or all water, i.e. at least 95% water removed,without disrupting the protein/antibody structure and intramolecularinteractions/functions. The proteins and/or antibodies are chemicallymodified into an ionic liquid, but when antibodies are used the modifiedantibodies maintain high antigen recognition, specificity, and bindingaffinity, e.g., the modified antibodies maintain picomolar (pM)dissociation constants (KD) about equal to those of native, unmodifiedantibodies. With regard to binding affinity, this means that theantigens bind strongly to the modified antibodies.

“Water-free” (as defined herein) protein liquids feature the simplicityof traditional inorganic ionic liquids (facile synthesis, ability totune properties through choice of cation and anion pair, and stability),but display the complexity and functionality of highly active proteins,e.g. antibodies. Because the protein liquids have most or all of thewater removed, they are stable liquids, resistant to extremetemperatures (>100° C.), able to maintain biological recognitionactivity, and exhibit much longer shelf-lives without the need forrefrigeration.

According to one embodiment of the present invention a method forcreating a stable protein ionic liquid, comprises: (a) cationizingaqueous proteins by addition of an excess of a positively-chargecrosslinker in the presence of a coupling agent; (b) purifying thecationized proteins; (c) titrating the cationized proteins with acorresponding biologically-compatible counter anionic polymer to createat least one antibody cation/anion pair in aqueous solution until theantibody cation/anion pair solution becomes negative by zeta potentialmeasurement; (d) dialyzing the at least one protein cation/anion pair inwater at least once to remove excess anionic polymer using at least onemolecular weight cutoff 7000 dialysis membrane; (e) lyophilizing the atleast one protein cation/anion pair to remove most of the water, forminga lyophilized solid; and (f) heating the lyophilized solid until aprotein ionic liquid is generated. In cationizing the aqueous proteins,a minimum zeta potential value of +5 mV is desired for cationization. Intitrating the cationized antibodies, the negative zeta potential ismeant below 0 mV to about −1 mV by zeta potential. A negative zetapotential of the titrated cationized antibodies ensures that there is aminor excess of anion but that the positive charges are equallybalanced. Heating of the lyophilized solid may be done on a hotplate, ina temperature controlled water bath, or an oven at about 27-50° C., forexample. This provides the advantage of producing stable,heat-resistant, biologically active protein ionic liquids that do notrequire refrigeration. In one embodiment of the present invention, theprotein ionic liquid is a viscous, clear liquid. Antibodies may includebut are not limited to IgG, IgY, IgM, and other proteins ornegatively-charged molecules may also be rendered stable according tothe teachings herein.

According to a first variation, the method for creating a stable proteinionic liquid further comprises purifying the cationized proteins fromexcess coupling reagents by dialysis in water. This provides theadvantage of obtaining a pure protein sample composed of only proteinsmodified with positive charges.

According to another variation, the method for creating a stable proteinionic liquid further comprises cationizing aqueous proteins by additionof an excess of or a stoichiometric amount ofN,N-dimethyl-1,3-propanediamine crosslinker in the presence of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling reagent.

According to a further variation, the method for creating a stableprotein ionic liquid further comprises cationizing aqueous proteins byaddition of an excess of or a stoichiometric amount of 2-(dimethylamino)ethanethiol crosslinker in the presence of succinimidyl iodoacetate(SIA) coupling agent.

According to a further variation, the method for creating a stableprotein ionic liquid further comprises cationizing aqueous proteins byaddition of an excess of or a stoichiometric amount of 2-(dimethylamino)ethanethiol crosslinker in the presence of N-(p-maleimidophenyl)isocyanate (PMPI) coupling agent.

According to another variation, the method for creating a stable proteinionic liquid further comprises performing the dialysis with at least onemembrane with a molecular weight cutoff (MWCO) of about 7000 g/mol.According to a further variation, the method for creating a stableprotein ionic liquid further comprises performing the dialysis with atleast one membrane with a molecular weight cutoff of between about6000-15,000 g/mol. In one embodiment this membrane may remove aplurality of contaminants and excess reagents from the modified proteinsthat are below a molecular weight, e.g. 7000 g/mol. A molecular weightof at least about 7000 g/mol typically ensures that all couplingreagents, positively-charged cross-linker, and buffer salts areseparated from cationized proteins. About 7000 g/mol may be the lowerlimits for this dialysis, however, the membrane could be as large as15,000 g/mol, but at the risk of losing proteins through the largermembrane.

According to a further variation, the method for creating a water-freeultra-stable protein ionic liquid further comprises confirming thecationizing of the aqueous proteins by measuring a positive zetapotential value. The zeta potential may be between about 0 mV and +5 mV.This provides the advantage of determining the number of positivecharges added to the protein.

According to another variation, the method for creating a stable proteinionic liquid further comprises titrating the cationized proteins withthe corresponding biologically-compatible counter anionic polymer ofpoly(ethylene glycol) 4-nonylphenyl 3-suopropyl ether, i.e.C₉H₁₉C₆H₄—(OCH₂CH₂)₂₀O(CH₂)₃SO₃. In other embodiments the counter anionpolymer may be biologically-derived DL-Lactate, biologically-derivedlinolenic acid, phospholipids, fatty acids, the conjugate base form ofamino acids (i.e. deprotonated and negatively charged), anybiologically-derived singly-charged anion with low melting points (e.g.between about 5-30° C.). This provides the advantage of balancing thepositive charges on the protein with negative charges of the anion toform the ionic salt form of the protein.

According to a further variation, the method for creating a stableprotein ionic liquid further comprises heating the lyophilized solid toabout 50° C. to generate the protein ionic liquid. This provides theadvantage of melting the protein ionic salt to form a viscous water-freeliquid without deactivating the protein.

According to another embodiment of the invention, the protein may be anantibody.

According to another variation, the method for creating a stable proteinionic liquid further comprises heating the protein ionic liquid at about100° C. for about 2 hours; and testing the protein ionic liquid forantibody recognition of a corresponding antigen, when the protein is anantibody. In one embodiment, the testing may be done using a dot blotassay on a nitrocellulose membrane. In a further embodiment, the heatingmay be between about 75° C. and about 150° C. and/or may be between 1and 3 hours. This provides the advantage of evaluating the temperaturestability of the protein/antibody ionic liquid at extreme temperaturesby directly measuring binding activity of the antibody for an antigen.

According to a further variation, the protein is an anti-hemoglobinantibody, polyclonal anti-horse spleen ferritin antibodies, monoclonalAnti-Flag antibodies, monoclonal Anti-HRP2 to Plasmodium falciparum,polyclonal Anti-neuropeptide Y, polyclonal Anti-human troponin, and allantibody isotypes, e.g. IgY, IgG, IgM, IgE, etc.

In addition, a dye, such as an IR active dye, may be combined withblood-typing antibody solutions via conjugation of an amine reactivedye, e.g., Anti-A ionic liquid, such that blood typing may beaccomplished without visible light using night vision goggles todetermine blood type via the hemagluttination of red blood cells, atremendous boon to soldiers and field medics in hazardous regions. Othervariations may be useful for lateral flow assays, enzyme-linkedimmunosorbent assays (ELISA), anti-venom/anti-toxin therapeutics,immunotherapy, vaccines, anti-virals, detection of chemical, biological,nuclear, environmental and radioactive agents, and may be applied toother biologically-important proteins whether negatively or positivelycharged, e.g., insulin.

Additional objects, advantages, and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the presentinvention and, together with a general description of the inventiongiven above, and the detailed description of the embodiments givenbelow, serve to explain the principles of the present invention.

FIG. 1 depicts a general approach to modify any protein or antibody intoa stable protein or antibody, according to an embodiment of the presentinvention.

FIG. 2 depicts a more-detailed approach to modify any protein orantibody into a stable protein or antibody, according to an embodimentof the present invention.

FIG. 3 depicts a typical protein or antibody that has been cationized,according to the present invention.

FIG. 4 depicts the cationization of a protein or antibody solution inthe presence of a coupling agent, according to an embodiment of thepresent invention.

FIGS. 5A-5B depict the selective cationization of IgG in the presence ofcoupling agents, according to an embodiment of the present invention.

FIG. 6 depicts the antigen binding of an aqueous antibody at roomtemperature and at 100° C., according to an embodiment of the presentinvention.

FIG. 7 depicts the antigen binding of an ionic liquid antibody at roomtemperature and at 200° C., according to an embodiment of the presentinvention.

It should be understood that the appended drawings are not necessarilyto scale, presenting a somewhat simplified representation of variousfeatures illustrative of the basic principles of the invention. Thespecific design features of the sequence of operations as disclosedherein, including, for example, specific dimensions, orientations,locations, and shapes of various illustrated components, will bedetermined in part by the particular intended application and useenvironment. Certain features of the illustrated embodiments have beenenlarged or distorted relative to others to facilitate visualization andclear understanding. In particular, thin features may be thickened, forexample, for clarity or illustration.

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate particular properties and advantagesof some of the embodiments of the present invention. Furthermore, theseare examples of reduction to practice of the present invention andconfirmation that the principles described in the present invention aretherefore valid but should not be construed as in any way limiting thescope of the invention.

This invention exploits the physical properties of ionic liquids and thebiological recognition of antigen-specific antibodies to create a stableand heat-resistant antibody protein ionic liquid that exhibitsrefrigeration-free storage and handling, which makes it suitable for useor storage at typical room temperatures. However, such a process hasnumerous obstacles to overcome because antibodies and many otherproteins are negatively charged. This makes such proteins and antibodiesdifficult to ionically combine with anions. In order to create an ionicliquid with antibodies, the antibodies' charge must be made morepositive. Antibodies have a great number of negative sites (e.g.carboxyl groups, —COOH; amine groups —NH₂; hydroxyl groups, —OH) toaddress, but in order to maintain the activity of the antibody thecationization process should not be too aggressive. In short, too fewpositive charges yields an antibody that does not function correctly asa salt. Too many positive charges yields an antibody with diminishedbiological activity, i.e. once the antibody's non-acid (general) aminoacids are coupled the antibody loses its specificity and its usefulness.

FIG. 1 depicts a general approach 10 to modify any antibody, e.g. anative antibody. In one embodiment, four solutions may be required toproduce an antibody ionic liquid: a solution of antibodies 12, asolution of cationic crosslinker molecules 14, a solution of couplingagents 16, and a corresponding anion 18. The anion 18 may bebiologically-derived or abiotic. The examples presented herein utilizebiologically-derived anions, but abiotic anions may be used in the samemanner. After the antibodies 12 are cationized (cationized antibodies 20with cationic crosslinker molecules depicted as “+”),biologically-derived (or biologically-compatible) anions 18 are combinedwith the cationized antibodies 20 to form an antibody/anion salt 22.Removal of all or most of the water, i.e. at least 95% or at least 99%,results in an ultra-stable antibody ionic liquid 24, which is depictedin a sample tube. The antibody ionic liquid 24 may require norefrigeration, may be stable at room temperature, and may be stable upto about 200° C.

FIG. 2 depicts another embodiment of the invention to modify anyantibody. Some of the numerous acidic sites, i.e., —COOH (carboxyl),basic sites —NH₂ (amine), and neutral sites —OH (hydroxyl) are depictedon a native (unmodified) antibody 30. At least some of the carboxyl,amine and hydroxyl groups may be modified in order to achieve a cationicantibody 32, i.e. the carboxyl, amine and hydroxyl groups (depicted onnative antibody 30) of the native antibody 30 are negative sites whichtend to make the native antibody 30 generally anionic. This may be doneselectively. Various cations may be used to selectively modify thecarboxyl, amine and hydroxyl groups. For example, EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) may be used to modifythe carboxyl groups, SIA (succinimidyl iodoacetate) may be used tomodify the amine groups, and PMPI (N-(p-maleimidophenyl) isocyanate) maybe used to modify the hydroxyl groups (not shown) to form an antibodycation 32. Cations in FIG. 32 are represented by “+”. If only a fractionof each carboxyl, amine and hydroxyl group is desired to be modified, inorder to maintain the functionality of the antibody salt, thestoichiometry may be adjusted to limit the reagents (e.g., EDC, SIA,PMPI) and thereby limit the number of groups, i.e. the carboxyl, amineand hydroxyl groups, which are modified. After the antibody iscationized 32, the cationized antibody 32 may be combined with an anion34 in order to form an antibody ionic liquid 36 after removal of most ofthe water. The antibody ionic liquid 36 is depicted in a sample tube 38.

FIG. 3 depicts a typical antibody 50 that has been cationized. A typicalantibody 50 has a constant region 52 and a variable region 54. Theconstant region 52 (corresponding to about the lower two-thirds of thedepicted antibody 50) is generally the same for antibodies. The variableregion 54, depicted as the upper ends of the Y branches, includes anantibody epitope 56 which will be distinct for each different type ofantibody, depending on its affinity for a specific antigen. This givesthe antigen its functionality. In one embodiment, only the constantregion 52 is modified so as to retain the functionality of the antigen50. Even with fewer than only about 5-15% of the amino acids in theconstant region 52 modified taking into account the total number ofamino acids in the constant region, or about 60-90% of the negativesites in the constant region, the resulting antibody ionic liquid willexhibit binding affinity and functionality with appropriate antigens.

FIG. 4 depicts the cationization of an antibody solution in the presenceof a coupling agent. In one embodiment, cationization gives the antibodya positive charge without neutralizing its functionality. Too fewpositive charges means the antibody will not form an ionic liquid withan anion. Too many positive charges may result in the antibody losingits functionality. FIG. 4 graphically illustrates how the concentrationsof the coupling agents may affect the overall charge of the antibodies,and accordingly, about how many anions will be bound with the cationicantibodies. There are about 144 acidic amino acids on a typicalantibody, and about 1600 total (acidic and non-acidic) amino acids. Thenon-acidic amino acids control the functionality of the antibodies. Iftoo many (more than about 30% of the total amino acids) non-acid aminoacids are coupled the antibody loses its specificity and affinity, i.e.it no longer functions as an antibody. The chart of FIG. 4 illustratesthat there is a practical limit as to how many anions may be bound by acationic antibody. The dashed line (line 63 with square data points)illustrates a cationic antibody solution that has been cationized atabout 10 equivalents (theoretic—about 10 positive charges per IgGantibody) based on the strength of the coupling agents. Line 63 startswith a negative zeta potential, which indicates that the cationizationwas insufficient to give the antibodies a positive charge overall. Thusthis low level of cationization is insufficient for use in making anantibody ionic liquid.

Line 62 (solid line with circle data points) illustrates a cationicantibody solution that has been cationized at about 100 equivalents(theoretic) based on the strength of the coupling agents. Line 62 startswith a positive zeta potential, which indicates that the cationizationwas sufficient to give the antibodies a positive charge overall. Thusthis level of cationization is sufficient for use in making an antibodyionic liquid. Likewise, line 61 (solid line with triangle data points)illustrates a cationic antibody solution that has been cationized atabout 1000 equivalents (theoretic) based on the strength of the couplingagents. Line 61 starts with a positive zeta potential, which indicatesthat the cationization was sufficient to give the antibodies a positivecharge overall. Thus this level of cationization is also sufficient foruse in making an antibody ionic liquid. However, the extra strength ofthe coupling agents did not affect the formation of the ionic liquid tothe degree expected from the concentration of the coupling agents.

FIG. 5A depicts another embodiment of the invention with the selectivecationization of immunoglobulin (IgG) in the presence of couplingagents. There are different numbers of the carboxyl, amine and hydroxylgroups in a typical antibody. These may be selectively coupled throughthe use of particular coupling agents, including, for example, SIA,PMPI, AMAS (N-α-maleimidoacet-oxysuccinimide ester), BMPS(N-β-maleimidopropyl-oxysuccinimide), SBAP (succinimidy3-(bromoacetamido) propionate), a photoactive coupling agent (e.g.ANB-NOS (N-5-azido-2-nitrobenzoylsuccinimide) or sulfo-SDA(sulfosuccinimidyl-4,4′-azipentanoate)), or BMPH (N-β-maleimidopropionicacid hydrazide), and combinations thereof. AMAS, BMPS or SBAP may beused as a substitute for SIA. For example, SIA may be used to cationizethe amine (—NH₂) sites, and PMPI may be used to cationize the hydroxyl(—OH) groups. Selective cationization of these groups in the antibodiesmay be accomplished with selected coupling agents, and/or the use ofselected coupling agents as limited reagents, in order to achieve adesired cationic state or positive zeta potential. Line 71 (line withsquare data points) corresponds to the cationization of carboxyl (—COOH)groups, line 72 (line with circular data points) corresponds to thecationization of amine (—NH) groups), and line 73 (line with triangulardata points) corresponds to the cationization of hydroxyl (—OH) groups).In one embodiment of the present invention, each of these groups may beselectively and/or partially cationized to achieve the desire cationicstate or zeta potential in order to function properly as an ionicliquid. FIG. 5B illustrates a comparison between the theoretical totalnumber of amino acid groups (—COOH or NH₂ or —OH) which may be modifiedwith a positive charge and the actual number that were modified in aparticular experiment. Out of a total of 144 —COOH groups (correspondingto line 1 of the graph presented on FIG. 5A), 115 of those weremodified, leaving 29 —COOH groups unmodified.

FIG. 6 depicts the antigen binding of an aqueous antibody at roomtemperature (i.e. about 21-25° C.) 81 and at 100° C. 82 using a quartzcrystal microbalance (QCM) to measure mass of antigen adsorbed to anantibody immobilized quartz sensor. FIG. 6 illustrates that an aqueousantibody solution cannot handle elevated temperatures, as is depicted bythe change in frequency response as temperature increases. As thetemperature increases to 100° C., the antibody solution exhibitsdecreasing binding activity until no binding activity is seen. That is,no binding is observed when a constant frequency value of 0 Hz+/−0.5 Hzover time is measured.

FIG. 7 depicts the antigen binding of an ionic liquid antibody at roomtemperature (i.e. about 21-25° C.) 91 and at 200° C. 92 using a quartzcrystal microbalance to measure mass of antigen adsorbed to an antibodyimmobilized quartz sensor. Binding is observed when the frequencydecreases by more than about 2 Hz over time and a clear slope isobserved vs. the initial baseline before antigen is added. FIG. 7illustrates that an ionic liquid antibody solution can handle elevatedtemperatures and maintains functionality, as is depicted by the changein frequency response as temperature increases. As the temperatureincreases to 200° C., the antibody ionic liquid continues to exhibitbinding activity similar to its performance at room temperature.

Numerous anions were identified as possibilities for making an antibodyliquid salt. However, non-biological polymer anions may trigger animmune response if used in vivo. A few biological anions were discoveredto be amenable to making protein ionic liquids, including DL-lactate,linolenate, phospholipids, fatty acids, and combinations thereof, whichare biocompatible. These are presented only as examples and theinvention is not intended to be limited solely to those biologicalanions. Any biologically-derived anion with a low melting point (e.g.between about 5-30° C.) that known in the art may be used. The samemethodology is generally applicable to all antibodies and yieldsantibody ionic liquids which are stable and maintain efficacy up to 200°C., as illustrated in FIG. 7.

In one embodiment, creation of a water-free ultra-stable antibody ionicliquid, aqueous anti-hemoglobin antibodies produced in rabbits werecationized by addition of stoichiometric amounts ofN,N-dimethyl-1,3-propanediamine in the presence of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling reagent;addition of succinimidyl iodoacetate (SIA) and 2-(dimethylamino)ethanethiol, and/or N-(p-maleimidophenyl) isocyanate (PMPI) and2-(dimethylamino) ethanethiol. After cationization, the cationizedantibodies were purified from excess coupling reagents by repeateddialysis in water using dialysis membranes with molecular weight cutoffs(MWCO) of 7000 g/mol. Cationized antibodies were confirmed by a positivezeta potential value.

Next, cationized antibodies were titrated with a corresponding non-toxicand bio-compatible counter anionic polymer ofC₉H₉C₆H₄—(OCH₂CH₂)₂₀O(CH₂)₃SO₃ until positive charges on antibody becameslightly negative by zeta potential measurements.

The antibody cation/anion pair was dialyzed repeatedly in water toremove excess anionic polymer using MWCO 7000 dialysis membranes andlyophilized to remove enough water.

Finally, lyophilized solid, e.g. powder, of the cationizedanti-hemoglobin/anion pair was slowly, e.g. over a 20-minute period ormore, heated to about 50° C. until a viscous clear liquid was generated.In one embodiment, the heating period is 30-90 minutes. In anotherembodiment, the cationized anti-hemoglobin pair is heated to 40-90° C.The anti-hemoglobin antibody ionic liquids were tested for antibodyrecognition of hemoglobin antigen using a dot blot assay on anitrocellulose membrane and after heating at about 100° C. for 2 hoursto test for temperature resistance. The antibody ionic liquid hadretained its functionality.

The resulting antibody ionic liquids are ultra-stable, possess longshelf-lives (i.e. greater than about 5 years), do not requirerefrigeration for storage/handling/use, do not have to adhere to a coldsupply chain, are resistant to extreme temperatures (such astemperatures greater than about 100° C.), are non-toxic and biologicallycompatible, and can be easily reconstituted into water or a biologicalbuffer for therapeutic use. By comparison, antibodies in aqueoussolutions have limited shelf-lives even with controlled refrigeration,are extremely sensitive to increased temperatures, and quickly lose allbiological recognition activity. In one embodiment, antibody ionicliquids provided by the disclosed method may reduce costs associatedwith refrigeration and may also eliminate the substantial weight burdenof heavy refrigeration equipment.

In one embodiment, water-free antibody liquids may also be prepared withstable single chain antibodies from camelids, antibody fragments, or maycontain combinations of multiple antibodies to create multi-recognitionantibody liquids.

Ultra-stable antibody liquids may permit refrigeration-free handling,storage and antibody-based diagnostics. They are resistant to extremetemperatures, have long shelf lives (e.g. a 20-fold improvement of theprior art), reduce the cost/weight load of specialized refrigerationequipment, and are able to be transported to underdeveloped countrieswhile maintaining efficacy.

While the present invention has been illustrated by a description of oneor more embodiments thereof and while these embodiments have beendescribed in considerable detail, they are not intended to restrict orin any way limit the scope of the appended claims to such detail.Additional advantages and modifications will readily appear to thoseskilled in the art. The invention in its broader aspects is thereforenot limited to the specific details, representative apparatus andmethod, and illustrative examples shown and described. Accordingly,departures may be made from such details without departing from thescope of the general inventive concept.

What is claimed is:
 1. A stable protein ionic liquid, comprising: ananti-hemoglobin cation/anion pair, wherein the anti-hemoglobincation/anion pair is: an anionic polymer of poly(ethylene glycol)4-nonylphenyl 3-sulfopropyl ether.
 2. The stable protein ionic liquid ofclaim 1, wherein the anti-hemoglobin cation/anion pair furthercomprises: a cationized anti-hemoglobin antibody, a single-chainantibody from camelids, antibody fragments, a polyclonal Anti-horsespleen ferritin antibody, a monoclonal Anti-Flag antibody, monoclonalAnti-HRP2 to Plasmodium falciparum, polyclonal Anti-neuropeptide Y,polyclonal Anti-human troponin, isotypes of antibodies, or a combinationthereof.